Use of ectoenzymes and secreted enzymes to monitor cellular proliferation

Abstract

The present invention relates to methods of measuring cellular proliferation using ectoenzymes such as membrane-bound chitobiase (N,N'-diacetylchitobiase) and nucleic acids for use in such methods.

What is claimed is:  
     
       1. A method for screening a test compound for the ability to inhibit microbial proliferation, said method comprising the steps of: 
       (a) providing a population of microbial cells expressing an ectoenzyme or secreted enzyme, wherein said population of cells is contacted with a sublethal level of an antisense nucleic acid that is complementary to at least a portion of a nucleic acid that encodes a gene product which is required for proliferation of said population of microbial cells, to reduce the activity or amount of said gene product in said cells, to thereby produce sensitized microbial cells;  
       (b) determining the extent of proliferation of said sensitized cells that express said ectoenzyme or secreted ezyme by measuring the activity of said ectoenzyme or secreted enzyme;  
       (c) contacting said sensitized cells with a test compound and measuring the extent of proliferation of said sensitized cells in response to said test compound; and  
       (d) determining whether said test compound inhibits the proliferation of said sensitized cells by comparing the activity of said ectoenzyme or secreted enzyme in said sensitized cells prior to contact with the test compound with the activity of said ectoenzyme or secreted enzyme following contact with the test compound.  
     
     
       2. The method of  claim 1 , wherein said ectoenzyme or secreted enzyme is selected from the group consisting of  Pseudomonas aeruginosa  metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase,  P. aeruginosa  FpvA ferric pyoverdin receptor,  E. coli  OmpP endopeptidase, outer membrane phospho lipase A,  Bacteriodes thetaiotamicron  susG starch utilization protein,  Haemophilus influenza ? phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase,  Lactococcus lactis  serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA,  S. pneumoniae  beta-N-acetylglucosaminidase,  S. pneumoniae  neuraminidase,  Streptococcus sobrinus  dextranase,  Streptococcus suis  muramidase,  Streptococcus mutans  exo-beta-D-fructosidase,  Staphylococcus aureus  murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase,  S. aureus  nuclease,  S. aureus  fatty acid modifying enzyme, chitinase,  S. aureus  autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. 
     
     
       3. The method of  claim 1 , wherein said ectoenzyme or secreted enzyme is a membrane-bound form of chitobiase. 
     
     
       4. The method of  claim 1 , wherein said ectoenzyme or secreted enzyme is endogenous. 
     
     
       5. The method of  claim 1 , wherein said sensitized cells contain an introduced gene encoding said ectoenzyme or secreted enzyme. 
     
     
       6. The method of  claim 1 , wherein said population of cells is from an organism selected from the group consisting of  Staphylococcus aureus, Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, E. coli, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Heisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum , and  Yersinia pestis  or any species falling within the genera of any of the above species. 
     
     
       7. The method of  claim 1 , wherein said antisense nucleic acid is transcribed from an inducible promoter. 
     
     
       8. The method of  claim 1 , further comprising the step of contacting said population of cells with a concentration of inducer which induces said antisense nucleic acid to a sublethal level. 
     
     
       9. The method of  claim 1 , wherein said sublethal level of the antisense nucleic acid is provided by contacting said population of cells with the antisense nucleic acid. 
     
     
       10. The method of  claim 1 , wherein said sublethal level of the antisense nucleic acid is provided by expressing the antisense in said population of cells. 
     
     
       11. The method of  claim 1 , wherein said gene product is a polypeptide. 
     
     
       12. The method of  claim 1 , wherein said gene product is an RNA. 
     
     
       13. The method of  claim 1 , wherein said test compound is from a combinatorial chemical library. 
     
     
       14. The method of  claim 1 , wherein said test compound is a natural product. 
     
     
       15. A method for screening a test compound for the ability to inhibit microbial proliferation, said method comprising the steps of: 
       (a) providing a first population of unsensitized microbial cells expressing an ectoenzyme or secreted enzyme, wherein said first population of cells is contacted with a sublethal level of an antisense nucleic acid that is complementary to at least a portion of a nucleic acid that encodes a gene product which is required for proliferation of said first population of microbial cells, to reduce the activity or amount of said gene product in said cells, to thereby produce sensitized microbial cells;  
       (b) determining the extent of proliferation of said sensitized cells that express said ectoenzyme or secreted enzyme by measuring the activity of said ectoenzyme or secreted enzyme;  
       (c) contacting said sensitized cells with a test compound and measuring the extent of proliferation of said sensitized cells in response to said test compound; and  
       (d) determining whether said test compound inhibits the proliferation of said sensitized cells by comparing the activity of said ectoenzyme or secreted enzyme in said sensitized cells prior to contact with the test compound with the activity of said ectoenzyme or secreted enzyme following contact with the test compound,  
       (e) providing a second population of unsensitized microbial cells, wherein said second population of unsensitized microbial cells are from the same population of microbial cells as said first population of unsensitized microbial cells, and said second population of unsensitized cells have not undergone sensitization treatment of any kind;  
       (f) determining the extent of proliferation for said unsensitized cells that express said ectoenzyme or secreted enzyme by measuring the activity of said ectoenzyme or secreted enzyme;  
       (g) contacting said unsensitized cells with a test compound and measuring the extent of proliferation of said unsensitized cells in response to said test compound; and  
       (h) determining whether said test compound inhibits the proliferation of said unsensitized cells by comparing the activity of said ectoenzyme or secreted enzyme in said unsensitized cells prior to contact with the test compound with the activity of said ectoenzyme or secreted enzyme following contact with the test compound;  
       (i) determining whether said test compound inhibits the proliferation of said sensitized cells to a greater extent than said compound inhibits the proliferation of said unsensitized cells by comparing the change in activity of said ectoenzyme or secreted enzyme in said sensitized cells following contact with the test compound with the change in activity of said ectoenzyme or secreted enzyme in said unsensitized cells following contact with the test compound.  
     
     
       16. The method of  claim 15 , wherein said ectoenzyme or secreted enzyme is selected from the group consisting of  Pseudomonas aeruginosa  metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase,  P. aeruginosa  FpvA ferric pyoverdin receptor,  E. coli  OmpP endopeptidase, outer membrane phospho ipase A,  Bacteriodes thetaiotamicron  susG starch utilization protein,  Haemophilus influenzae  phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase,  Lactococcus lactis  serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA,  S. pneumoniae  beta-N-acetylglucosaminidase,  S. pneumoniae  neuraminidase,  Streptococcus sobrinus  dextranase,  Streptococcus suis  muramidase,  Streptococcus mutans  exo-beta-D-fructosidase,  Staphylococcus aureus  murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase,  S. aureus  nuclease,  S. aureus  fatty acid modifying enzyme, chitinase,  S. aureus  autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase, and enterotoxin. 
     
     
       17. The method of  claim 15 , wherein said ectoenzyme or secreted enzyme is a membrane-bound form of chitobiase. 
     
     
       18. The method of  claim 15 , wherein said ectoenzyme or secreted enzyme is endogenous. 
     
     
       19. The method of  claim 15 , wherein said sensitized cells contain an introduced gene encoding said ectoenzyme or secrete enzyme. 
     
     
       20. The method of  claim 15 , wherein said population of cells is from an organism selected from the group consisting of  Staphylococcus aureus, Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, E. coli, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum , and  Yersinia pestis  or any species falling within the genera of any of the above species. 
     
     
       21. The method of  claim 15 , wherein said antisense nucleic acid is transcribed from an inducible promoter. 
     
     
       22. The method of  claim 15 , further comprising the step of contacting said first population of unsensitized cells with a concentration of inducer which induces said antisense nucleic acid to a sublethal level. 
     
     
       23. The method of  claim 15 , wherein said sublethal level of the antisense nucleic acid is provided by contacting said first population of unsensitized cells with the antisense nucleic acid. 
     
     
       24. The method of  claim 15 , wherein said sublethal level of the antisense nucleic aid is provided by expressing the antisense in said first population of unsensitized cells. 
     
     
       25. The method of  claim 15 , wherein said gene product is a polypeptide. 
     
     
       26. The method of  claim 15 , said gene product is an RNA. 
     
     
       27. The method of  claim 15 , wherein said test compound is from a combinatorial chemical library. 
     
     
       28. The method of  claim 15 , wherein said test compound is a natural product.